Tuberc Respir Dis.  2010 Jul;69(1):16-23.

Induction of Autophagy by Low Dose of Cisplatin in H460 Lung Cancer Cells

• Shin JH
• Jang HY
• Chung JS
• Cho KH
• Hwang KE
• Kim SY
• Kim HJ
• Lee SY
• Lee MK
• Park SA
• Moon SR
• Lee KK
• Jo HJ
• Yang SH

Abstract

BACKGROUND
Most lung cancer patients receive systemic chemotherapy at an advanced stage disease. Cisplatin-based chemotherapy is the main regimen for treating advanced lung cancer. Recently, autophagy has become an important mechanism of cellular adaptation under starvation or cell oxidative stress. The purpose of this study was to determine whether or not autophagy can occurred in cisplatin-treated lung cancer cells.
METHODS
H460 cells were incubated with RPMI 1640 and treated in 5 micrometer or 20 micrometer cisplatin concentrations at specific time intervals. Cells surviving cisplatin treatment were measured and compared using an MTT cell viability assay to cells that underwent apoptosis with autophagy by nuclear staining, apoptotic or autophagic related proteins, and autophagic vacuoles. The development of acidic vascular organelles was using acridine orange staining and fluorescent expression of GFP-LC3 protein in its transfected cells was observed to evaluate autophagy.
RESULTS
Lung cancer cells treated with 5 micrometer cisplatin-treated were less sensitive to cell death than 20 micrometer cisplatin-treated cells in a time-dependent manner. Nuclear fragmentation at 5 micrometer was not detected, even though it was discovered at 20 micrometer. Poly (ADP-ribose) polymerase cleavages were not detected in 5 micrometer within 24 hours. Massive vacuolization in the cytoplasm of 5 micrometer treated cells were observed. Acridine orange stain-positive cells was increased according in time-dependence manner. The autophagosome-incorporated LC3 II protein expression was increased in 5 micrometer treated cells, but was not detected in 20 micrometer treated cells. The expression of GFP-LC3 were increased in 5 micrometer treated cells in a time-dependent manner.
CONCLUSION
The induction of autophagy occurred in 5 micrometer dose of cisplatin-treated lung cancer cells.

Keyword

Autophagy; Cisplatin; Lung Neoplasms

MeSH Terms

Acridine Orange
Apoptosis
Autophagy
Cell Death
Cell Survival
Cisplatin
Cytoplasm
Humans
Lung
Lung Neoplasms
Organelles
Oxidative Stress
Proteins
Starvation
Vacuoles
Acridine Orange
Cisplatin
Proteins

Figure

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  Figure 1 Effect of cisplatin-induced cell death on H460 cells. Tumor cells were seeded at 5×104 cells per well in 48-well flat-bottomed plates and incubated overnight at 37℃. After treated with cisplatin (5 µM or 20 µM) for 3 hr, 6 hr, 12 hr, 24 hr, 48 hr, and 72 hr, the viability of the untreated cells was regarded as 100%. The viability of cell was measured by MTT assay. Control: no cisplatin-treated cells. Means±SD (n=3). *p≤0.05, †p≤0.01.

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  Figure 2 Cell death induced by cisplatin in NCI-H460 cells did not involve apoptosis. Cells were treated with 5 µM, 20 µM or 5 µM cisplatin with 10 mM 3-MA for 24 hr and assayed for apoptosis by FACS following annexin V-FITC staining. Control cells were treated with RPMI 1640 medium. The data shown are representative of three independent experiments.

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  Figure 3 Lung cancer cells were treated for several times with 5 µM and 20 µM cisplatin. Western blot analysis showing effects of cisplatin. Western immunoblots probed with antibodies against poly (ADP-ribose) polymerase (PARP) & cleaved PARP and Actin.

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  Figure 4 Cisplatin induced morphologic changes of H460 cells. Direct observation of cells treated with 5 µM and 20 µM cisplatin for 2 hr, 6 hr, 12 hr, 24 hr, and 48 hr. Scale bar: ×200. Sample were examined under a light microscope.

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  Figure 5 AVOs development in cisplatin-treated H460 cells and its quantificanion using FACS analysis. H460 cells contained RPMI 1640 (control), 5 µM, 20 µM cisplatin or 3-MA with 5 µM cisplatin for 24 hr were stained with acridine orange and analyzed by fluorescent microscopy. Representative images in each group were shown. Note the formation of acridine orange-accumulating AVOs (bright red fluorescence) in cisplatin-treated H460 cells. Similar results were obtained in three independent experiments. *p≤0.01.

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  Figure 6 Induction of autophagic protein LC3-I and formation of LC3-II in H460 cells. Cells were treated with 5 µM, 20 µM cisplatin for various time periods as indicated. LC3-I (18 kDa) and LC3-II (16 kDa) induction and conversion of LC3-I to LC3-II were determined by Western blotting.

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